Use of —furin—“convertase” inhibitors in the treatment of fibrosis and scarring

ABSTRACT

The present invention relates to use of convertase inhibitors for the reduction of scarring during the healing of wounds and also for reducing fibrosis in the treatment of fibrotic conditions.

This application claims priority under 35 U.S.C. § 119 toPCT/GB2003/003159, filed 23 Jul. 2003, which designates the U.S. andwhich claims priority to Great Britain Application 0217136.1, filed 24Jul. 2002, which are incorporated herein by reference.

The present invention relates to wound healing and also to regulatingfibrosis in the treatment of conditions in which fibrosis is a majormechanism of tissue repair or where excessive fibrosis leads topathological derangement and malfunctioning of tissue.

Wound healing in adults is a complicated reparative process. The term“wound” as used herein is exemplified by, but not limited to, injuriesto the skin. Other types of wound can involve damage, injury or traumato an internal tissue or organ such as the lung, kidney, heart, gut,tendons or liver.

The healing process in skin wounds typically begins with a haemostaticresponse initiated by damage to blood vessels in the skin. During thisprocess platelets and a number of factors present in the bloodcontribute to the formation of a clot that prevents further blood loss.Factors released during this process, particularly by the degranulationof platelets, then cause recruitment of a variety of specialised cellsto the site of the wound that are in turn involved in extracellularmatrix and basement membrane deposition, angiogenesis, selectiveprotease activity and re-epithelialisation. An important component ofthe healing process in adult mammals is the stimulation of fibroblaststo generate the extracellular matrix. This extracellular matrixconstitutes a major component of the connective tissue that develops torepair the wound area.

The connective tissue that forms during the healing process is oftenfibrous in nature and commonly forms into a connective tissue scar (aprocess known as fibrosis).

A scar is an abnormal morphological structure resulting from a previousinjury or wound (e.g. an incision, excision or trauma). Scars arecomposed of a connective tissue which is predominately a matrix ofcollagen types 1 and 3 and fibronectin. The scar may consist of collagenfibres with an abnormal organisation (as seen in scars of the skin) orit may be an abnormal accumulation of connective tissue (as seen inscars of the central nervous system). Most scars consist of abnormallyorganised collagen and also excess collagen. In man, in the skin, scarsmay be depressed below the surface or elevated above the surface of theskin. Hypertrophic scars represent a severe form of normal scarring.They are elevated above the normal surface of the skin and containexcessive collagen arranged in an abnormal pattern. Keloids are anotherform of pathological scarring in which the scar is not only elevatedabove the surface of the skin but also extends beyond the boundaries ofthe original injury. In a keloid there is excessive connective tissuethat is organised in an abnormal fashion predominately in whirls ofcollagenous tissue. There are genetic predispositions to the formationof both hypertrophic scars and keloids. These aberrant forms of scarringare particularly common in Afro-Caribbean and Mongoloid races.

There are many instances where the regulation of scar formation is ofprimary importance when considering the outcome of wound healing.Examples of such situations are scars of the skin where excessivescarring may be detrimental to tissue function, particularly in contextswhere scar contracture occurs (for instance skin burns and wounds thatimpair flexibility of a joint). The reduction of scarring to the skinwhen cosmetic considerations are important is also highly desirable. Inthe skin hypertrophic or keloid scars can cause functional and cosmeticimpairment and there is a need to prevent their occurrence. Scarringresulting from skin grafts in both donor sites and from the applicationof artificial skin can also be problematic and need to be minimised orprevented.

As well as scars of the skin, internal scarring or fibrosis can behighly detrimental and specific examples include:

-   -   (i) Within the central nervous system, glial scarring can        prevent neuronal reconnection (e.g. following neuro-surgery or        penetrating injuries of the brain).    -   (ii) Scarring in the eye can be detrimental. In the cornea,        scarring can result in abnormal opacity and lead to problems        with vision or even blindness. In the retina, scarring can cause        buckling or retinal detachment and consequently blindness.        Scarring following wound healing in operations to relieve        pressure in glaucoma (e.g. glaucoma filtration surgery) results        in the failure of the surgery whereby the aqueous humour fails        to drain and hence the glaucoma returns.    -   (iii) Scarring in the heart (e.g. following surgery or        myocardial infarction) can give rise to abnormal cardiac        function.    -   (iv) Operations involving the abdomen or pelvis often result in        adhesion between viscera. For instance, adhesions between        elements of the gut and the body wall may form and cause        twisting in the bowel loop leading to ischaemia, gangrene and        the necessity for emergency treatment (untreated they may even        be fatal). Likewise, trauma or incisions to the guts can lead to        scarring and scar contracture to strictures which cause        occlusion of the lumen of the guts which again can be life        threatening.    -   (v) Scarring in the pelvis in the region of the fallopian tubes        can lead to infertility.    -   (vi) Scarring following injury to muscles can result in abnormal        contraction and hence poor muscular function.    -   (vii) Scarring or fibrosis following injury to tendons and        ligaments can result in serious loss of function.

Related to the above is the fact that there are a number of medicalconditions known as fibrotic disorders in which excessive fibrosis leadsto pathological derangement and malfunctioning of tissue. Fibroticdisorders are characterised by the accumulation of fibrous tissue(predominately collagens) in an abnormal fashion within the tissue.Accumulation of such fibrous tissues may result from a variety ofdisease processes. These diseases do not necessarily have to be causedby surgery, traumatic injury or wounding. Fibrotic disorders are usuallychronic. Examples of fibrotic disorders include cirrhosis of the liver,liver fibrosis, glomerulonephritis, pulmonary fibrosis, scleroderma,myocardial fibrosis, fibrosis following myocardial infarction, centralnervous system fibrosis following a stroke or neuro-degenerativedisorders (e.g. Alzheimer's Disease), proliferative vitreoretinopathy(PVR), restenosis (for example following angioplasty) and arthritis.There is therefore also a need for medicaments which may be used for thetreatment of such conditions by regulating (i.e. preventing, inhibitingor reversing) fibrosis/scarring in these fibrotic disorders.

Whilst the above considerations mainly apply to conditions, disorders ordiseases of man it will be appreciated that wound healing, scarring andfibrotic disorders can also be problematic in other animals,particularly veterinary or domestic animals (e.g. horses, cattle, dogs,cats etc). For instance abdominal wounds or adhesions are a major reasonfor having to put down horses (particularly race horses), as are tendonand ligament damage leading to scarring or fibrosis.

There have been several recent developments in the fields of woundhealing, scarring and fibrotic disorders. Some of these developmentsrevolve around the recent understanding that an array of cytokines andgrowth factors is intimately involved in the repair of wounded tissue.In particular, members of the Transforming Growth Factor β (TGF-β)superfamily have been found to play an important role in wound healing.At least 25 molecules are known to be members of the TGF-β superfamily.These include a number of cytokines such as TGF-βs 1 to 5, the DVR group(e.g. dpp and Vg1), Bone Morphogenetic Proteins, Nodal, Activin andInhibin.

TGF-βs are often secreted from cells in an inactive form known as latentTGF-β. Latent TGF-β consists of an N terminal Latency Associated Peptide(LAP) and the TGF-β and is also referred to as the Small Latent Complex.Additionally the Small Latent Complex can bind to another peptide(derived from a different gene) of variable size called Latent TGF-βBinding Protein (LTBP) in which case the entire complex is known as theLarge Latent TGF-β Complex.

Latent TGF-β is activated when the TGF-β is caused to be dissociatedfrom the LAP. This dissociation may be coordinated at amannose-6-phosphate/Insulin Like Growth Factor II receptor (M6P-R) andinvolve proteases such as plasmin, the substrates being associated atthe cell surface by tissue transglutaminase. Free radicals and reactiveoxygen species can also activate TGF-β by causing dissociation from theLAP.

TGF-β (particularly TGF-β₁ and TGF-β₂) promotes wound healing but isalso associated with increased scar formation and fibrosis. Clinicalinterest in the modulation of TGF-β has been associated with inhibitingits activity in order to reduce scar formation (although this maycompromise the rate of wound healing). For instance, WO 92/17206discloses neutralising agents that inhibit the activity of TGF-β₁ andTGF-β₂ and are particularly beneficial for reducing scar formation.

Another development involves the use of mannose-6-phosphate for use intreating fibrotic disorders associated with accumulation ofextracellular matrix and with elevated levels of Transforming GrowthFactors β₁ or β₂ (GB-A-2,265,310). Mannose-6-phosphate is believed tointerfere with the conversion of latent forms of these TransformingGrowth Factors into their active form.

Despite such advances there remains a need to continue to developmedicaments that may be used to modulate the healing of wounds, scarringand fibrosis. In particular there is a need for medicaments which do notcompromise the rate of wound healing or quality of scar in favour of oneor the other.

As discussed more fully below, the invention relates in its broadestaspect to the use of convertase inhibitors for the treatment of wounds.

According to a first aspect of the present invention there is providedthe use of a convertase inhibitor in the manufacture of a medicament forreducing scarring during the healing of wounds or reducing fibrosis inthe treatment of fibrotic conditions wherein the medicament is topicallyapplied to the site of a wound or fibrotic disorder.

According to a second aspect of the present invention, there is provideda composition comprising a therapeutically effective amount of aconvertase inhibitor and a pharmaceutically acceptable vehicle for thetreatment of wounds or fibrosis.

According to a third aspect of the present invention, there is provideda method of treating a subject to reduce or prevent scarring during thehealing of wounds; or reduce or prevent fibrosis in the treatment offibrotic conditions comprising topically administering to a subject inneed of such treatment a therapeutically effective amount of aconvertase inhibitor.

Convertases are a family of Ca²⁺-dependant serine proteases, otherwiseknown as SPCs (subtilisin-like pro-protein convertases; see Dubois etal., 1995, Journ. Biol. Chem., 270(18):10618-10624; Sha, X., et al.,1989, Mol. Endocrinology, 3:1090-1098; Chan, S. J., et al., 1992, Proc.Natl. Acad. Sci. USA 89: 6678-6682; and references therein). Theinventors have found that the convertase enzyme furin is particularlyinvolved in the activation of mature latent TGF-β at the site of a woundor fibrotic disorder. Although the inventors do not wish to be bound byany hypothesis they believe that convertase activity is able toindirectly stimulate TGF-β activation by modifying the activity of otherenzyme(s) with TGF-β activating properties. The inventors believe thatthe convertase activity contributing to TGF-β activation initiallyoccurs intracellularly, within the platelet, and then continuesextracellularly as the platelet contents are released on de-granulation.Accordingly convertase inhibitors used according to the presentinvention are believed to modify activity of this enzyme such that TGF-βactivation is reduced.

For the purposes of the specification references to intracellularactivity should also be taken to encompass activity within the membranesof cell fragments, such as platelets, except where the context requiresotherwise.

The inventors believe that the convertases involved in TGF-β activationare furin-like proprotein convertases. Furins comprise a family of seventransmembrane proprotein convertases produced as an inactive precursor.They must be activated intracellularly, and are involved in pre-proteinprocessing in the trans-Golgi network, at the cell surface,extracellularly and in endosomes. Furins have their effect atarginine-containing cleavage sites, the minimal site being Arg-X-X-Arg.Relevant reviews include Molloy et al, 1999; Shapiro et al. 1997 (J.Histochem. Cytochem. 45:3-12) and Pearton et al. 2001 (ExperimentalDermatology 10:193-203).

Platelets are a major source of TGF-β in the circulation and releaselatent TGF-β when the platelet is activated (e.g. in response toinjury). During the healing process, various forms of TGF-β are to befound at a wound site or site of a fibrotic disorder. These differentforms are active TGF-β (which is in its free form), the small latentcomplex (TGF-β-latency associated peptide), and the large latent complex(TGF-β-latency associated peptide-latent TGF-β binding protein). Thedifferent complexes undergo different fates and perform different rolesduring the healing process. In particular, the large and small latentcomplexes are activated by cleaving in order to release active TGF-βwhilst the healing process occurs.

The prior art suggests that cleavage of the large and small latentcomplexes at a wound site is mediated by plasmin. Furthermore,convertases such as furin are believed to be responsible for theintracellular processing of pro-TGF-β within megakaryocytes (which giverise to platelets) in the bone marrow. This processing of pro-TGF-βinvolves cleavage and folding of the pro-protein to produce the matureform. The mature form produced is not, however, “active” TGF-β, and maybe associated with the large or small latent complexes. Accordinglyconvertases have not previously been thought to play a part in theactivation of latent TGF-β (such as TGF-β in the small latent complex)from platelets in the blood at a site of a wound or fibrotic disorder.

However, the inventors have established (as described in more detail inthe Example) that, surprisingly, activity of convertase enzymes such asfurin effects the extracellular activation of TGF-β at a wound site.Hence by inhibiting the activity of convertase enzymes at a wound siteor site of a fibrotic disorder it is possible to reduce the amount ofactive TGF-β at such a site and thereby reduce scarring and/or fibrosis.It is interesting to note that this activity of convertases appears tooccur intracellularly and extracellularly. Furthermore the activity doesnot appear to be associated with, or controlled by, transcriptionalregulation (in contrast to known convertase activity in cells such asmegakaryocytes) and thus is able to take place in the anuclear platelet,or even extracellularly.

The novel observation that convertase enzymes are involved in activationof TGF-β is in contrast to the previously reported role of these enzymesin TGF-β processing and maturation, and opens a range of therapeuticpossibilities that could not have been envisaged before.

Although the prior art recognises that platelets contain latent TGF-β itprovides no indication that it is possible to prevent activation of thisTGF-β by inhibiting convertase activity.

Instead the prior art only suggests that inhibitors of convertase areable to inhibit the processing and maturation that produces latentTGF-β. This activity of convertases is transcriptionally regulated. Thusthe skilled person would have recognised that once latent TGF-β ispresent in circulating platelets convertase inhibitors would not be ableto influence its state.

It is only with the present invention that it can be seen thatconvertase inhibitors are able to prevent the activation, andundesirable effects, of this platelet-borne latent TGF-β at a woundsite.

The efficacy of convertase inhibitors for reducing scarring or fibrosisis enhanced by the fact that the latent TGF-β released by platelets isalmost entirely composed of TGF-β₁ (associated with the small latentcomplex). It is well known that TGF-β₁ is a key factor in wound healingand is pro-fibrotic favouring scar formation. The preponderance of thepro-fibrotic TGF-β isoforms during the early phases of wound healing(which causes local conditions that favour scar formation and fibrosis)is in large measure due to platelet-mediated growth factor release. Withtime the ratio of TGF-β isoforms changes as levels of platelet-derivedTGF-β₁ decrease and there is an increase in the levels of anti-fibroticTGF-β₃ derived from fibroblasts. Preventing the activation of latentTGF-β₁ according to the present invention can therefore dramaticallyreduce the degree of scarring associated with wound healing.

Several classes of compound may be used according to the invention asconvertase inhibitors. These compounds include:

-   -   (i) compounds that bind to convertase enzymes and inhibit its        activity (e.g. competitive inhibitors or allosteric inhibitors);    -   (ii) compounds which prevent the transcription, translation or        expression of convertase enzymes (e.g. ribozymes or antisense        DNA molecules);    -   (iii) compounds which increase the rate of degradation of        convertase enzymes;    -   (iv) compounds which inhibit the interaction of convertase        enzymes with latent TGF-β and/or with TGF-β activating proteins;    -   (v) compounds which inhibit the proteolytic activation of the        inactive furin precursor; and    -   (vi) compounds which inhibit a potential intracellular        translocation of convertase enzymes, such as furin or PACE-4, to        subcellular sites of activity.

In one embodiment of the invention it is preferred that the convertaseinhibitor is an inhibitor of furin.

In a further embodiment of the invention it is preferred that theconvertase inhibitor is an inhibitor of furin-like proproteinconvertases, and more preferably an inhibitor of PACE-4.

The convertase inhibitor may be a serine protease inhibitor and ispreferably a thiol inhibitor. The thiol inhibitor may be a peptidylchloroalkylketone having a peptide moiety which mimics at least oneconvertase enzyme cleavage site. It has been found that peptidylchloroalkylketones with peptide moieties that mimic the convertaseenzyme cleavage site are specific inhibitors of the enzymatic activity.A preferred inhibitor is decanoyl-RVKR-cmk (SEQ ID NO:1) and derivativesthereof.

Further convertase inhibitors suitable for use according to theinvention include:

-   (i) alpha 1-antitrypsin (α-1 PDX), or nucleic acids encoding the    same;-   (ii) derivatives of alpha 1-antitrypsin such as those comprising the    amino acid sequences arg-val-pro-arg (SEQ ID NO: 4), ala-val-arg-arg    (SEQ ID NO: 5) or arg-val-arg-arg (SEQ ID NO: 6), or nucleic acids    encoding the same;-   (iii) p-chloromercuribenzoate;-   (iv) tosylamido-phenylethyl chloromethyl ketone (TPCK);-   (v) D-polyarginines (e.g. hexa-arginine (SEQ ID NO: 7) and its    derivatives);-   (vi) Acetyl-leu-leu-arg-aldehyde hemisulfate;-   (vii) S-carboxyphenylethyl-carbamoyl-arg-val-arg-aldehyde;-   (viii) Threodimercaptobutanediol; and-   (ix) Tos-Lys-chloromethylketone.

Alternatively and/or in addition, the convertase inhibitor may sequesterCa²⁺. Furthermore a Ca2+ sequester (such as EDTA or EGTA) may be used inconjunction with inhibitors such as those mentioned above.

The inventors have established that convertase enzymes act, bothextracellularly and intracellularly, to cause the activation of latentTGF-β in the extracellular space at the site of a wound or a fibroticcondition. This led them to realise that it is possible to useconvertase inhibitors applied topically to prevent the activation oflatent TGF-β associated with wound healing or fibrosis. The prior artsuggests that the activity of convertases such as furin takes place inthe megakaryocytes in the bone marrow that give rise to platelets, andthat this activity is limited to the processing that produces latentTGF-β. Accordingly the prior art contains no teaching that suggests thatlocal inhibition of furin activity would inhibit the conversion oflatent TGF-β to active TGF-β. Furthermore the prior art suggest thattherapeutic manipulation of furin would require an agent that may bedelivered to, and achieve its action in, the bone marrow.

The surprising finding that extracellular convertase activitycontributes to TGF-β activation led the inventors to realise thatwater-soluble convertase inhibitors may be used to decrease TGF-βactivation, and thus reduce scarring. The use of water-solubleinhibitors, which cannot penetrate the cell membrane, is particularlyadvantageous since such inhibitors generally exhibit low levels ofcytotoxicity. Water-soluble convertase inhibitors can also be readilyformulated into compositions that induce minimal inflammatory reactions,an important consideration when designing anti-scarring agents. Apreferred water-soluble convertase inhibitor suitable for use accordingto the invention is L-hexaarginine (SEQ ID NO: 7).

The effects of localised inhibition of convertase activity are verydifferent from those that would arise as a result of systemicadministration of convertase inhibitors. Even were a skilled person tosuggest that systemic use of convertase inhibitors would indirectlyreduce levels of active TGF-β by inhibiting processing of pro-TGF-β inthe bone marrow they would also understand that such an approach wouldhave a number of deleterious effects. One such effect would be thatsystemic administration of convertase inhibitors would also reduce thelevel of anti-fibrotic TGF-β₃ that is important in the later stages ofwound healing. A second problem is that systemic use of convertaseinhibitors would have detrimental, and possibly toxic, effects sinceconvertases are involved in the normal processing of proteins other thanTGF-β.

Neither of these disadvantages is applicable to the topical applicationof convertase inhibitors according to the invention. In this use theeffect of the inhibitors is directed to the region of the wound, or siteof fibrosis, and the administration of the inhibitors may be timed sothat only the initial quantities of TGF-β released from platelets areaffected.

As set out above, the inventors believe that the convertase activitycontributing to TGF-β activation is initiated within the platelets, andthat the extracellular convertase activity occurs as a result ofconvertases being released into the extracellular space onde-granulation of the platelets. As a result, inhibitors of convertaseactivity for use according to the invention may be inhibitors that areable to cross the cell membrane and act intracellularly. Such inhibitorsare able to reduce convertase activity prior to de-granulation. They maybe able to decrease TGF-β activation occurring both intracellularly andextracellularly. That said, it appears that the majority of convertaseactivity contributing to TGF-β activation occurs extracellularly afterplatelet degranulation. Thus water-soluble inhibitors may be used toeffectively reduce TGF-β activation and thus reduce scarring and/orfibrosis.

In a preferred embodiment of the invention the medicament containing theconvertase inhibitor may be applied prophylactically. Thus themedicament may be applied to a site where a wound may be formed orfibrosis may occur (e.g. before elective surgery).

The inventors find that the inhibitors according to the presentinvention are highly suited for topical application to dermal wounds ordermal fibrotic conditions.

Convertase inhibitors used according to the invention may be proteins orhave peptidyl components. Such proteins can easily be modified (forinstance by amino acid addition, substitution or deletion) to formderivatives that retain the ability to inhibit enzymes such as film.Therefore derivatives that retain functional characteristics ofnaturally occurring proteins are also preferred inhibitors of theinvention. Examples of such derivatives include functionally activefragments of naturally occurring proteins and even precursors ofnaturally occurring proteins (e.g. proproteins) which are activated insitu.

Convertase inhibitors may be used according to the invention insituations or conditions where scarring needs to be prevented or reducedsuch as:

(i) where scars of the skin may be excessive and/or detrimental totissue function and particularly when scar contracture occurs or mayoccur (for instance skin burns and wounds which impair flexibility of ajoint and particularly scarring in children);

(ii) scarring to the skin when cosmetic considerations are important;

(iii) when hypertrophic or keloid scars particularly in Afro-Caribbeanand Mongoloid races) may occur which can cause functional and cosmeticimpairment;

(iv) scarring resulting from skin grafts in both donor sites and fromthe application of artificial skin;

(v) scarring within the central nervous system (e.g. followingneuro-surgery or penetrating injuries of the brain), for example glialscarring can prevent reconnection of severed neurons;

(vi) scarring in the eye and particularly of the cornea (scarring canresult in abnormal opacity and lead to problems with vision or evenblindness), in the retina (scarring can cause buckling or retinaldetachment and consequently blindness) and scarring following woundhealing in operations to relieve pressure in glaucoma (e.g. glaucomafiltration surgery) which can result in the failure of the surgerywhereby the aqueous humour fails to drain and hence the glaucomareturns;

(vii) scarring in the heart (e.g. following surgery or myocardialinfarction) which can give rise to abnormal cardiac function;

(viii) scarring of the gut such as may occur following operationsinvolving the abdomen or pelvis that result in adhesion between viscera(adhesions between elements of the gut and the body wall can form andcause twisting in the bowel loop leading to ischaemia, gangrene and thenecessity for emergency treatment-untreated they may even be fatal);likewise, trauma or incisions to the guts can lead to scarring and scarcontracture or strictures which cause occlusion of the lumen of the gutswhich again can be life threatening;

(ix) scarring in the pelvis in the region of the fallopian tubes whichcan lead to infertility,

(x) scarring following injury to muscles which can result in abnormalcontraction and hence poor muscular function;

(xi) scarring or fibrosis following injury to tendons and ligamentswhich can result in serious loss of function.

The convertase inhibitors may also be used for the treatment orprevention of fibrosis. For instance the compounds may be used to treatfibrotic disorders such as cirrhosis of the liver, liver fibrosis,glomerulonephritis, pulmonary fibrosis, scleroderma, myocardialhibernation, fibrosis following myocardial infarction, central nervoussystem fibrosis following a stroke or neuro-degenerative disorders (e.g.Alzheimer's Disease), proliferative vitreoretinopathy (PVR), restenosisand arthritis.

The convertase inhibitors are useful for reducing or preventing fibrosisin fibrotic diseases and for reducing or preventing the formation offibrosis that manifests as hypertrophic scarring (particularly of theskin) or keloids.

Wound healing compositions used according to the invention may take anumber of different forms depending, in particular on the manner inwhich they are to be used. Thus, for example, they may be in the form ofa liquid, ointment, cream, gel, hydrogel, powder or aerosol. All of suchcompositions are suitable for topical application to skin, which is apreferred means of administering convertase inhibitors to a subject(person or animal) in need of treatment.

The convertase inhibitors may be provided on a sterile dressing or patchwhich may be used to cover or even pack a wound to be treated.

A preferred composition of the invention may be in the form of aninjectable solution (e.g. for intradermal injection around the marginsof a wound or a site to be wounded).

It will be appreciated that the vehicle of the composition of theinvention should be one which is well tolerated by the patient andallows release of the active convertase inhibitor to the wound. Such avehicle is preferably biodegradeable, bioresolveable and/ornon-inflammatory.

The composition of the invention may be used in a number of ways. Thus,for example, a composition may be applied in, and/or around a wound of apatient to regulate wound healing. If the composition is to be appliedto an “existing” wound, then the pharmaceutically acceptable vehiclewill be one which is relatively “mild” i.e. a vehicle which isbiocompatible, biodegradable, bioresolvable and non-inflammatory.

It is also possible to use compositions in accordance with the inventionprior to surgery (particularly elective surgery) so as to provide forregulation of healing of the subsequently formed surgical wound. In thiscase the vehicle of the topically applied composition may need to be onecapable of going across the keratinous layer of the skin. Examples ofsuitable vehicles for this purpose include dimethyl sulphoxide andacetic acid. Such prophylactic use is a preferred use of convertaseinhibitors according to the invention.

The compositions are suitable to be used for reducing or controllingscarring resulting form surgical operations on the eye (e.g. lasersurgery on the cornea). In this case the composition or medicament maybe in the form of an eye drop.

Convertase inhibitors may be used in a range of internal wound healingapplications. Thus for example, the composition may be formulated forinhalation for use in wound healing of the lungs or for the preventionor treatment of fibrosis and strictures in the lung.

It will be appreciated that the amount of a convertase inhibitor to beapplied to the wound site depends on a number of factors such as thebiological activity and bioavailability of the compound, which in turndepends on the mode of administration and the physicochemical propertiesof the inhibitor. Other factors may include:

A) The half-life of the inhibitor in the subject being treated.

B) The specific condition to be treated.

C) The age of the subject.

The frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the convertaseinhibitor within the subject being treated.

Generally when the compositions are used to treat existing wounds orfibrotic disorders the convertase inhibitor should be administered assoon as the wound has occurred or the disorder has been diagnosed.Therapy with the composition should continue until the wound has healedto a clinician's satisfaction or, in the case of a fibrotic disorder,the risk or cause of abnormal fibrous tissue formation has been removed.

Compositions which modulate scarring and/or fibrotic disorders shouldalso be applied to a wound as soon as possible after the wound hasformed. However scars and fibrosis can develop over days or even weeks.Therefore the subject being treated may well benefit by administrationof a convertase inhibitor even if it is administered days or even weeksafter the wound occurred or the disorder developed (or was diagnosed).

When used as a prophylactic (e.g. before surgery or when there is a riskof developing a fibrotic disorder) the convertase inhibitors should beadministered as soon as the risk of undesirable fibrosis has beenrecognised. For instance, a cream or ointment containing a convertaseinhibitor may be applied to a site on the skin of a subject whereelective surgery is to be performed and decreased scar formation issubsequently desired. In this case, the composition may be appliedduring the preoperative preparation of the subject or it may even bedesirable to apply the composition in the hours or days preceding thesurgery (depending upon the health status and age of subject as well asthe size of the wound to be formed).

Frequency of administration will depend upon the biological half-life ofthe inhibitor used. Typically a cream or ointment containing aconvertase inhibitor should be administered to a target tissue such thatthe concentration of the inhibitor at the wound site or tissue affectedby a fibrotic condition is maintained at a level suitable for having atherapeutic effect. This may require administration daily or evenseveral times daily.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g. in vivo experimentation, clinical trialsetc), may be used to establish specific formulations of compositions andprecise therapeutic regimes (such as daily doses of the convertaseinhibitor and the frequency of administration).

Generally, compositions for use in accordance with the invention shouldbe formulated such that when administered to a wound site or site of afibrotic disorder that a convertase inhibitor concentration of between0.01 μM and 10 mM is achieved at the site.

Purely by way of example an injectable solution containing between 0.1.mu.M and 10 mM of decanoyl-RVKR-cmk (SEQ ID NO:1) is suitable forapplication to an existing (i.e. “open”) wound.

A suitable daily dose of a compound which inhibits convertase activitydepends upon the factors discussed above as well as upon the size of thewound, or amount of tissue effected by fibrosis, which is to be treated.Typically the amount of a convertase inhibitor required for thetreatment of wounds or fibrotic disorders will be within the range of0.01 μg to 100 mg of the active compound/24 hours depending upon thesize of the wound or extent of fibrosis amongst several other factors.

It will also be appreciated that convertase inhibitors may be isolatedfrom nature or chemically synthesised.

Many known methods of administering convertase inhibitors to a relevanttissue have the disadvantage that it can be difficult to achievesustained levels of the active convertase inhibitor at a wound site orsite of fibrosis over the course of even a few days because convertaseinhibitors may have short half-lives in vivo. The half-lives of theconvertase inhibitors may be short for a number of reasons whichinclude:

(i) Degradation by proteases and the like.

(ii) Clearance by binding proteins (e.g. α2 macroglobulin).

(iii) Binding and inhibition of agent activity by extracellular matrixmolecules such as decorin and fibronectin.

Furthermore, compounds for wound healing and/or treatment ofscarring/fibrosis need to be administered in a suitable vehicle and areoften provided as a composition comprising the compound and the vehicle.As outlined above, such vehicles are preferably non-inflammatory,biocompatible, bioresorbable and must not degrade or inactivate theactive compound (in storage or in use). However, it can often bedifficult to provide a satisfactory vehicle for delivering specificcompounds to a tissue to be treated.

A convenient way in which these problems can be obviated or mitigated isto provide at a wound site (or site of fibrosis) a therapeuticallyeffective amount of protein or peptide convertase inhibitor by genetherapy.

According to a fourth aspect of the present invention there is provideda delivery system for use in a gene therapy technique, said deliverysystem comprising a DNA molecule encoding for a protein which inhibitsconvertase activity, said DNA molecule being capable of beingtranscribed to lead to the expression of said protein.

According to a fifth aspect of the present invention there is providedthe use of a delivery system as defined in the preceding paragraph foruse in the manufacture of a medicament for use in the treatment ofwounds or fibrosis.

According to a sixth aspect of the present invention there is provided amethod of treating a wounds or fibrosis comprising administering to apatient in need of treatment a therapeutically effective amount of adelivery system as defined for the fifth aspect of the invention.

The delivery systems according to the invention are highly suitable forachieving sustained levels of a convertase inhibitor at a wound site orsite of fibrosis over a longer period of time than is possible for mostconventional delivery systems. Protein may be continuously expressedfrom cells at the wound site or site of fibrosis that have beentransformed with the DNA molecule of the invention. Therefore, even ifthe protein has a very short half-life as an agent in vivo,therapeutically effective amounts of the protein may be continuouslyexpressed from the treated tissue.

Furthermore, the delivery system of the invention may be used to providethe DNA molecule (and thereby the protein which is an active therapeuticagent) without the need to use conventional pharmaceutical vehicles suchas those required in ointments or creams that are contacted with thewound.

The delivery system of the present invention is such that the DNAmolecule is capable of being expressed (when the delivery system isadministered to a patient) to produce a protein which directly orindirectly has activity for wound healing and/or treatment of fibrosisor scarring by inhibiting convertase activity. By “directly” we meanthat the product of gene expression per se has the required activity forwound healing and/or regulating fibrosis or scarring. By “indirectly” wemean that the product of gene expression undergoes or mediates (e.g. asan enzyme) at least one further reaction to provide an agent effectivefor wound healing and/or regulating fibrosis or scarring by inhibitingconvertase activity.

The DNA molecule may be contained within a suitable vector to form arecombinant vector. The vector may for example be a plasmid, cosmid orphage. Such recombinant vectors are highly useful in the deliverysystems of the invention for transforming cells with the DNA molecule.

Recombinant vectors may also include other functional elements. Forinstance, recombinant vectors may be designed such that the vector willautonomously replicate in the nucleus of the cell. In this case,elements which induce DNA replication may be required in the recombinantvector. Alternatively the recombinant vector may be designed such thatthe vector and recombinant DNA molecule integrates into the genome of acell. In this case DNA sequences which favour targeted integration (e.g.by homologous recombination) are desirable. Recombinant vectors may alsohave DNA coding for genes that may be used as selectable markers in thecloning process.

The recombinant vector may also further comprise a promoter or regulatorto control expression of the gene as required.

The DNA molecule may (but not necessarily) be one which becomesincorporated in the DNA of cells of the subject being treated.Undifferentiated cells may be stably transformed leading to theproduction of genetically modified daughter cells. When this is thecase, regulation of expression in the subject may be required e.g. withspecific transcription factors, gene activators or more preferably withinducible promoters which transcribe the gene in response to a signalspecifically found at a wound site. Alternatively, the delivery systemmay be designed to favour unstable or transient transformation ofdifferentiated cells in the subject being treated. In this instance,regulation of expression may be less important because expression of theDNA molecule will stop when the transformed cells die or stop expressingthe protein (ideally when the wound, fibrosis or scarring has beentreated or prevented).

The delivery system may provide the DNA molecule to the subject withoutit being incorporated in a vector. For instance, the DNA molecule may beincorporated within a liposome or virus particle. Alternatively the“naked” DNA molecule may be inserted into a subject's cells by asuitable means e.g. direct endocytotic uptake.

The DNA molecule may be transferred to the cells of a subject to betreated by transfection, infection, microinjection, cell fusion,protoplast fusion or ballistic bombardment. For example, transfer may beby ballistic transfection with coated gold particles, liposomescontaining the DNA molecule, viral vectors (e.g. adenovirus) and meansof providing direct DNA uptake (e.g. endocytosis) by application ofplasmid DNA directly to the wounded area topically or by injection.

The protein expressed from the DNA molecule may be one which directly orindirectly provides for wound healing with reduced scarring or one whichserves to regulate (inhibit, prevent or reverse) fibrosis.

It will be appreciated that the delivery system according to the fifthaspect of the invention may be used according to the sixth or seventhaspects of the invention to treat any of the conditions hereinbeforedescribed.

The present invention will further be described in the followingnon-limiting Example which refers to the accompanying drawing, in which:

FIG. 1 illustrates the results of analysis of the ability of differentinhibitors, and putative inhibitors, of TGF-β activation to preventTGF-β activation by platelets. ‘LSKL’ is disclosed as SEQ ID NO: 8 and‘dec-RVKR-cmk’ is disclosed as SEQ ID NO: 1;

FIG. 2 illustrates the effect of Dec-RVKR-cmk (SEQ ID NO:1) andhexaarginine (SEQ ID NO: 7) in: A platelet releasates; and Bplatelet-free releasates as referred to in experimental results section2 of the example. In A ▪ indicates active hexaarginine (SEQ ID NO: 7)and □ indicates total hexaarginine (SEQ ID NO: 7) whereas indicatesactive dec-RVKR-cmk (SEQ ID NO:1) and ◯ indicates total dec-KR RVKR-cmk(SEQ ID NO:2). In panel B ◯ indicates hexaarginine (SEQ ID NO: 7)whereas ● indicates dec-RVKR-cmk (SEQ ID NO:1); and

FIG. 3 illustrates the effect of furin inhibitors on furin activity incell lysates and releasates for control samples (▪); dec-RVKR-cmk (SEQID NO:1) (

); and hexaarginine (SEQ ID NO: 7) (□) in experimental results section 2of the example.

EXAMPLE

The ability of inhibitors of furin activity to inhibit activatedplatelets' production of active TGF-β was demonstrated by the ability ofthe inhibitors to abrogate the platelet's release and activation ofTGF-β in response to stimulation with thrombin. The experimentalprotocol was as set out below.

1. Protocols.

Collection and Preparation of Human Platelets:

Peripheral venous blood samples from healthy adult volunteers (aged 21to 45) were taken into 20-gauge S-Monovettes. EDTA was used to preventcoagulation of the samples. Human platelets were isolated bydifferential centrifugation according to the following protocol:

Blood samples were centrifuged at 300 g for 10 minutes to produce asupernatant of platelet rich plasma. Platelet rich plasma was thencentrifuged at 2,500 g for 15 minutes to produce a pellet of platelets.Platelets were harvested from the pellet and resuspended prior to use.

Production of Platelet Hypotonic Lysates:

Platelets collected as described above were resuspended in distilledwater and incubated at room temperature for 5 minutes. Lysis was stoppedby addition of an equal volume of 2× serum-free DME medium containing0.2% pyrogen poor bovine serum albumen. The lysate was cleared bycentrifugation at 12,000 g for 10 minutes at room temperature beforeuse.

2. Experimental Results 1.

2.1. Platelets Activated with Thrombin Release and Activate TGF-β:

Human platelets collected as outlined above were activated by theaddition of thrombin (0.1 u/ml for 30 minutes at 37° C.). Thisactivation caused the release of large amounts of TGF-β from theplatelets (total TGF-β60.2 ng/ml average), and also triggered thegeneration of significant amounts of active TGF-β (159 pg/ml on averageif the platelets were static during activation, 896 pg/ml on average ifthe platelets were subjected to agitation during activation) in theplatelet releasates as assayed using the PAI/L bioassay for TGF-βdescribed by Abe et al. (“An assay for transforming growth factor-βusing cells transfected with a plasminogen activator inhibitor-1promoter-luciferase construct” 1994, Anal. Biochem. 216: 276-284).Pan-specific neutralising anti-TGF-β antibodies completely abolished thesignal obtained in the PAI/L assay verifying that active TGF-β wasmeasured. Activation of latent TGF-β was platelet-mediated, sinceexogenous thrombin added to platelet releasates was unable to activatelatent TGF-β directly (data not shown). Antibody inhibition experimentsconfirmed that platelets expressed exclusively TGF-β₁ (data not shown).

2.2. Platelets Contain Intracellular Active TGF-β:

The presence of active TGF-β within platelets was confirmed by bothimmuno-localisation and bioassay studies.

Confocal microscope immuno-fluorescence studies of the localisation ofactive TGF-β within platelets were carried out using the chicken derivedactive TGF-β1-specific IgY AF-101-NA (R&D Systems) and an antibodyspecifically reactive with the membrane marker CD41 (BD Biosciences).Subcellular localisation of these proteins was investigated in opticalsections collected at 0.5 μm intervals along the z-axis. TGF-β1 wasdemonstrated to be present, and to have an intracellular localisation(lying within that of the membrane marker CD41).

The PAI/L bioassay for TGF-β was used to validate the presence of activeTGF-β in platelets through assaying for the presence of active TGF-β inhypotonic lysates of human platelets (prepared as described above).

In lysates derived from resting platelets 99% of the total TGF-β present(total TGF-β 34.7 ng/ml) was the latent form, although a significantproportion of active TGF-β (104 pg/ml) was detected. In lysates derivedfrom thrombin activated platelets the mean total TGF-β was 19.5 μg/ml,of which 151 pg/ml was active.

The results of the bioassay thereby confirmed the immuno-localisationfinding that platelets contain active TGF-β.

2.3. Inhibition of Furin Activity Inhibits TGF-β Activation by ThrombinActivated Platelets:

In a comparative experiment the ability of inhibitors of knownactivators of TGF-β (such as TSP-1, plasmin, M6P/IGF-II receptor) andputative activators of latent TGF-β such as other serine proteinases,cysteine proteinases, calpain I and II, caspase-3, and furin) to blocklatent TGF-β activation in human platelets was assessed.

Human platelets were pre-incubated with the inhibitors (listed below)prior to stimulation with thrombin (as above), and active and totalTGF-β levels were determined in the platelet releasates using the PAI/Lassay (as above). The results are shown in FIG. 1, panels A to C.

The results showed that latent TGF-β activation was not significantlyaffected by the presence of inhibitors specific for TSP-1 (LSKL peptide(SEQ ID NO: 8)), plasmin (neutralising monoclonal antibody, PG19—aneutralising antibody provided by Dr. Michael Kramer), or M6P/IGF-IIreceptor (M6P-mannose-6-phosphate) (panel A). Moreover, a number ofinhibitors (aprotinin, pefabloc, α1-antitrypsin, E-64, pepstatin,leupeptin, caspase-3 inhibitor and calpain inhibitor) of otherproteinases potentially involved in platelet-mediated latent TGF-βactivation also proved to be ineffective (panel B).

In comparison platelet incubation with a membrane-permeable inhibitor offurin-like proprotein convertases, dec-RVKR-cmk (SEQ ID NO:1)(decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-Bachem), drasticallyreduced the generation of active TGF-.beta. in releasates as well asintracellularly in a dose-dependent fashion (panel C). (intracellularmeasurements were taken from hypotonic lysates). The inventors believethat the residual TGF-.beta. present (approximately 20-30%) wasactivated prematurely during platelet preparation prior to the additionof the inhibitor.

2.4. Summary.

The above results indicate that platelet-mediated latent TGF-βactivation surprisingly occurs extracellularly at the site of plateletactivation (i.e. a wound site or a site of a fibrotic condition) andinvolves proteolytic processing by a furin-like convertase enzyme. Theyfurther show that the activation of latent TGF-β by platelets can besuccessfully inhibited by treatment of the platelets with an inhibitorof furin activity which may be applied topically.

As skilled person will appreciate from these results that convertaseinhibitors may be used to inhibit TGF-β₁ activation and will thereby beeffective as anti-scarring or anti-fibrotic agents.

3. Experimental Results 2.

3.1. Furin-Like Enzymes are Involved in Platelet-Mediated LatentTGF-Activation.

Platelets were activated with thrombin in the absence or presence offurin inhibitors. Platelets were pre-incubated with hexaarginine (SEQ IDNO: 7), whereas dec-RVKR-cmk (SEQ ID NO:1) was added 5 mm after thrombinaddition because of interference with platelet activation at higherconcentrations. Active and total TGF-β in releasates were determined inthe PAI/L bioassay. The results are shown in panel A of FIG. 2. ActiveTGF-β levels in the controls were 82 pg/ml (dec-RVKR-cmk (SEQ ID NO:1)data) and 61 pg/ml (hexaarginine (SEQ ID NO: 7) data), total TGF-βlevels were 33.4 ng/ml (dec-RVKR-cmk (SEQ ID NO:1) data) and 39.5 ng/ml(hexaarginine (SEQ ID NO: 7) data).

In a further experiment, furin inhibitors were added to platelet-freereleasates of activated platelets, and activation was allowed tocontinue in the absence of platelets for 30 mm at 37° C. The results ofthis experiment are shown in panel B of FIG. 2. Active TGF-β levels inthe controls were 77 pg/ml (dec-RVKR-cmk data (SEQ ID NO:1)) and 104pg/ml (hexaarginine data (SEQ ID NO: 7)). Incubation on ice reducedactivation in the controls to approximately 56% (data not shown). Datarepresent the mean values of three independent experiments assayed intriplicate.

The results illustrate that incubation of thrombin-stimulated plateletswith the membrane-permeable protease inhibitor, dec-RVKR-cmk (SEQ IDNO:1), considerably reduces the generation of active TGF-.beta. inreleasates (FIG. 2 panel A). Dec-RVKR-cmk (SEQ ID NO:1) is a specificand potent inhibitor of subtilisin/Kex2p-like proprotein convertases,with its peptide sequence being based on the substrate recognitionsequence of these enzymes.

The most prominent and ubiquitously expressed member of thisendoprotease family is furin, which typically cleaves at the consensussequence motif R-X-(K/R)-R. The membrane-impermeable furin inhibitor,hexa-L-arginine (SEQ ID NO: 7), also significantly reduced active TGF-βin releasates (FIG. 2 panel A) indicating that at least part of theactivation occurred extracellularly following latent TGF-β release.

Latent TGF-β activation appeared to be enzymatic and independent of thecontinuous presence of platelets, since incubation of platelet-freereleasates on ice (as compared to 37° C.) reduced active TGF-β levels toapproximately 56%. As observed for platelet suspensions, activation inreleasates was inhibited, in a dose-dependent fashion, by the furininhibitors, dec-RVKR-cmk (SEQ ID NO:1) and hexa-L-arginine (SEQ ID NO:7) (FIG. 2B). This indicates that the furin-like enzyme involved inlatent TGF-β activation is released from activated platelets.

3.2. Platelets Contain and release Furin-Like Enzyme Activity.

Releasates or hypotonic lysates of activated platelets were assayedusing the furin substrate, pyr-RTKR-amc (SEQ ID NO:3) in the absence orpresence of the furin inhibitors, hexaarginine (SEQ ID NO: 7) (200 μM)or dec-RVKR-cmk (SEQ ID NO:1) (150 μM). Values were corrected forsubstrate-independent endogenous fluorescence (control withoutsubstrate) as well as for spontaneous substrate hydrolysis (buffercontrol). Mean values±S.E.M. of 2-3 separate experiments assayed induplicate are shown.

The presence of furin-like enzyme activity in both hypotonic lysates andreleasates of human platelets was analysed using the fluorogenic furinsubstrate, pyr-RTKR-amc (SEQ ID NO:3). Platelet lysates contained afurin-like enzyme activity, part of which (approximately 12%) wasreleased upon thrombin stimulation. Enzyme activity in cell lysates andreleasates was inhibited by dec-RVKR-cmk (SEQ ID NO:1) andhexa-L-arginine (SEQ ID NO: 7) (FIG. 3).

The extracellular generation of active TGF-β by thrombin-stimulatedhuman platelets was significantly reduced in the presence of inhibitorsof furin-like proprotein convertases.

3.3 Summary.

Furin-like proprotein convertases catalyze the maturation of pro-TGF-βprecursor to heat-activatable latent growth factor complex. Our dataindicate, however, that platelet β-granules contain and release mature,heat-activatable latent TGF-β, and that the levels are not affected byfurin inhibitors. Thus, pro-TGF-β processing in the megakaryocyticlineage occurs in the megakaryocytes. These data therefore identify anovel function of furin-like enzymes, namely involvement in theextracellular activation of platelet large latent TGF-β₁ complex underphysiological conditions.

In summary, the inventors found that platelets are not only majorstorage sites for latent TGF-β1 but also activate part of it followingdegranulation. While the mechanism of activation does not require any ofthe well-characterized activators, TSP-1, M6P/IGF-II receptor, orplasmin, the platelet latent TGF-β complex appears to be activated via asequence of events by a furin-like convertase released by the platelets.Following release in vivo, this enzyme appears to continue to operate,independently of the presence of platelets, in the surrounding tissue(e.g. the wound area), leading to the activation of extracellular-matrixassociated latent TGF-β complex. Therefore, this novel mechanism ofactivation represents a target to modulate TGF-β activity in pathologicconditions involving platelet degranulation, such as wound repair,fibrosis, arteriosclerosis, and cancer. Therefore the inventors havefound that inhibitors according to the invention (such asdecanoyl-RVKR-cmk (SEQ ID NO:1) and hexa-arginine (SEQ ID NO: 7) may beused according to the invention).

1. A method for reducing scar formation during the healing of at leastone wound in a subject who has incurred injuries that result in said atleast one wound or in a subject who will be inflicted with at least onesurgical wound at a known site comprising applying a furin inhibitor,wherein the furin inhibitor is decanoyl-RVKR-cmk, directly to the siteof said wound.
 2. The method of claim 1 wherein said injury is to amuscle, tendon, ligament or skin.
 3. The method of claim 1 wherein thewound is a surgical wound.
 4. The method of claim 1 wherein saidapplying is topical.
 5. The method of claim 1 wherein said applying isby injection.